Presentation Summary

Written by Milica Maksimovic
Reviewed by John Andrew Sayer

Cystinuria is a genetic renal stone disease distinguished by the mutations in genes SLC3A1 and SLC7A9 encoding the proteins that facilitate dibasic amino acid exchange in the gut and the proximal tubule.

The disease is characterised by urinary loss of dibasic amino acids: cysteine, ornithine, lysine, and arginine. Because cystine is a homodimer of the amino acid cysteine, cystinuria is diagnosed by a decreased proximal tubular reabsorption of filtered cystine and excessive amount of urinary cystine, which leads to the formation of recurrent urinary stones and an increased risk of chronic renal impairment. The disease can be presented at any age but the median age of onset is twelve years. Modern diagnostic methods employ the combination of molecular genetic testing, analysis of 24-hour urine and its examination by atomic force microscopy, and evaluation of stone composition.

Molecular Genetics of Cystinuria

Heterodimeric amino acid transporter accountable for the absorption of cystine in the renal proximal tubule consists of rBAT and b0,+AT subunits, joined by disulfide bridge and encoded by SLC3A1 and SLC7A9 genes, respectively. Biallelic mutation in SLC3A1 denotes genotype AA, and a heterozygous one identify genotype A. These genotypes are typically inherited as autosomal-recessive. The biallelic change in SLC7A9 leads to genotype BB, whereas a heterozygous one to genotype B. These genotypes are inherited as both autosomal-recessive and autosomal-dominant with incomplete penetrance pattern. Due to digenic inheritance of two or more mutant alleles, rarer forms do exist, including type AB, type ABB and type AAB cystinuria.

Recently, the attention has been given to a gene SLC7A13 that encodes the protein AGT1 working alongside rBAT in the S3 section of proximal tubule but it remains to be seen how prevalent these mutations are in cystinuria patients.

First-line Treatment Approaches

Whereas the normal cystine urine elimination is 0.13 mmol per day, the cystinuria patients excrete more than 1.7 mmol per day. Because cystine is insoluble in urine, it firstly starts to form crystals, followed by stones once concentration exceeds its solubility level. The limit of solubility is reached at around 1.0 mmol per litre at a urine pH 7, and it considerably increases as the urine becomes alkaline. Importantly, supersaturation can occur at different points during the day or night.

Cystinuria involves lifelong treatment. The cystine stones are very resistant to typical therapies, therefore one of the therapeutic approaches includes minimisation of the amount of cystine in the urine by increasing the fluid intake, by dietary modification as well as by alkalinisation of the urine.

Maintaining the high fluid intake, especially during the night and before bed is aiming to produce at least three litres of urine per day. The therapeutic target is to keep the cystine concentration of less than 1 mmol/litre but it is essential to determine how much cystine each patient is excreting. Mobile phone applications may be used to prompt patients to drink water at regular intervals. Regarding the dietary modifications, patients are advised to follow moderate sodium diet that minimises excess urinary cystine excretion, as well as low animal protein consumption in order to alkalinise the urine and to reduce the intake of the amino acid methionine. Alkalinisation therapy maintains the urinary pH of cystinuria patients from 7.0 to 7.5 and facilitates the production of small smooth or finely rough stones (Figure 1). Potassium citrate and potassium bicarbonate supplements are given to patients, the doses of which are measured based on the individual urine pH. It is advised that the patients buy a pH meter so that they could regularly check own urine pH levels

Figure 1. Two morphological types of cystine kidney stones: a) granular with blunted edges (left) and b) regularly shaped with smooth appearance after treatment with alkali or/and thiol therapy (right) (slides 18 and 19 [1,2]).

Cystine-Binding Thiol Drugs

Cystine-binding thiol drugs (CBTDs) are another approach in treating cystinuria. Two main CBTDs, tiopronin and D-penicillamine, bind to cystine in the urine, reduce the disulfide bond and ultimately produce drug-bound cysteine complexes, which are up to 50 times more soluble than cystine. There are tolerability issues with both drugs so the patients developing proteinuria, anaemia, leucopenia, and liver dysfunction need to be monitored. Cystine capacity (CysCap) is a novel assay developed by Litholink (Chicago, Illinois, United States of America) for the prediction of kidney stone events in cystinuria. The solid-phase assay measures amounts of cystine crystals after incubation of urine samples for 48 hours with a pre-formed amount of cystine crystals. In supersaturated urine, the recovered solid phase is greater than the one that was added. Such urine has a ‘negative cystine capacity (CysCap)’. A ’Positive CysCap’ urine can dissolve the added cystine crystals. A patient with a positive CysCap has less stone events.

The CysCap test is used to judge the effectiveness of CBTD tiopronin. The tiopronin dose-response study showed that the CysCap increased in tiopronin treated patients when the dose of 1g per day was applied. However, there was no consistent further increase in CysCap or in cystine excretion within 24 h with higher tiopronin doses [3].

In addition to CysCap test, crystalluria and crystal volume are identified by microscopic examination of urine sediment. The retrospective study, performed in order to identify therapeutics targets using cystine crystalluria as a marker of cystine stone development, showed that a decrease in urine specific gravity substantially decreased the risk of cystine crystalluria [4].

Chaperone Therapy, Crystal Inhibitors and Other Approaches

Chaperone therapy is an attractive treatment for cystinuria since it is already applied on several mutations affecting the rBAT protein that cause protein misfolding.

Specially designed synthetic inhibitors of crystal growth are known to modulate crystallisation in urine, and could be able to slow or prevent cystine stones. As the stones are aggregates of individual hexagonal crystals and they generate hillocks in a spiral growth pattern, it is shown that L-cystine dimethyl ester (L-CDME) and L-cystine methyl ester (L-CME) significantly decrease the growth velocity of hillocks steps. L-CDME inhibitor affects the roughening of the crystal step edges in a higher rate than L-CME. L-CDME is indicated to be a viable therapeutic agent for the prevention of L-cystine stones [5]. In order to assess the efficacy of L-CDME as an inhibitor of crystal growth, the Slc3a1 knockout mice were given either water or L-CDME for four weeks. The treatment with L-CDME led to formation of high number of small stones but it did not prevent stone formation. Ultimately, the study showed that L-CDME could be used as an applicable treatment for cystine stones without any negative effects on the mice [6].

Other approaches include the vasopressin V2 receptor antagonist (V2RA) tolvaptan and the application of CRISPR/Cas9 gene editing systems. Tolvaptan causes polyuria, with urine output that averages six litres per day and as such is an attractive pharmacotherapeutic for cystine stones [7]. In an effort to apply gene therapy to repair SLC7A9 mutation by delivery of CRISPR/Cas9 reagents, there are numerous barriers to be overcome.


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